Antiviral phosphonate compounds and methods therefor

ABSTRACT

Pharmaceutical compositions comprise a nucleotide analog with a phosphonate group at a concentration effective to act as a substrate and/or inhibitor of a viral polymerase, and especially of the HCV RNA dependent RNA polymerase.

[0001] This application claims the benefit of U.S. provisionalapplication No. 60/377,024, filed Apr. 30, 2002, which is incorporatedherein by reference.

FIELD OF THE INVENTION

[0002] The field of the invention is pharmaceuticals, and especiallythose comprising nucleotide analogs.

BACKGROUND OF THE INVENTION

[0003] HCV infection poses a significant public health problem worldwideand is recognized as the major cause of non-A, non-B hepatitis. Theetiologic agent of hepatitis C, the hepatitis C virus (HCV) is widelyregarded as a member of the Flaviviridae family and category ofarboviruses, having a single-stranded positive-sense RNA genome encodinga polyprotein of approximately 3000 amino acids.

[0004] Although HCV infection resolves itself in some cases, the virusestablishes chronic infection in up to 80% of the infected individuals,and may persist for decades. It is estimated that about 20% of theseinfected individuals will go on to develop cirrhosis, and 1 to 5% willdevelop liver failure and hepatocellular carcinoma (Seeff, et al. 1999,Am. J. Med. 107:10S-15S; Saito, et al. 1990, Proc. Natl. Acad. Sci. USA,87:6547-6549; WHO, 1996, Weekly Epidemiol. Res., 71:346-349). Chronichepatitis C is the leading cause of chronic liver disease, and theleading indication for liver transplantation in the United States. TheCenters for Disease Control and Prevention estimate that hepatitis C iscurrently responsible for approximately 8,000 to 10,000 deaths in theUnited States annually. This number is projected to increasesignificantly over the next decade. Unfortunately, development ofeffective and safe vaccines for HCV has been impeded by the relativelyhigh immune evasion, probably due to a comparably high degree ofheterogeneity of this virus. Still further, mechanistic studies of HCVreplication have been hindered by the lack of an in vitro cell culturesystem and a validated small animal model as an alternative to thechimpanzee.

[0005] Currently, the most widely prescribed HCV antiviral therapy isthe combination of interferon-alpha 2b (IFN-alpha-2b, Intron A) andribavirin, a treatment capable of suppressing viral titers in about 40%of chronically infected patients. However, immunogenicity and relativelylow serum half-life times of interferon-alpha 2b have limited treatmentsuccess, at least in some patients. To overcome at least some of thedifficulties with immunogenicity and relatively low serum half-lifetime, interferon-alpha 2b has been coupled to polyethylene glycol. Useof such modified (pegylated) interferon in HCV therapy has significantlyimproved the clinical outcome for numerous patients. However, there isstill a significant portion of patients in which viral propagationpersists, even when Ribavirin is coadministered with pegylatedinterferon.

[0006] Alternative proposed and experimental pharmaceutical compositionsfor treatment of HCV infections include various nucleoside analogs.Exemplary compositions are described in WO 01/90121 to Novirio, WO02/57425 to Merck, or WO 02/100354 to Ribapharm. While some of thesecompositions may have an antiviral effect to at least some degree,various disadvantages remain. Among other things, selectivity of suchcompounds for the viral polymerase may be less than desirable. Otherpotential disadvantages may include poor phosphorylation to thecorresponding nucleotide (or nucleotide analog).

[0007] Furthermore, certain phosphonate nucleoside analogs are known toexhibit significant and selective antiviral effect. For example,9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a potent antiviral agentagainst the hepatitis B virus (see e.g., U.S. Pat. Nos. 4,659,825,4,724,233 or 4,808,716). However, PMEA is also extremely toxic andtherefore has failed to provide a viable drug. In another example,3′-Azido-3′,5′-dideoxythymidine-5′-methylphosphonic acid diphosphateshowed promising antiviral effects against the human immunodeficiencyvirus (HIV), but exhibited relatively poor selectivity and is generallydifficult to administer, specifically to infected cells (see e.g., J.Med. Chem. 1992 Aug. 21;.35(17):3192-6). Still further, Watanabe et al.describe in published U.S. Patent Application US 20020055483 selected2′,3′-substituted nucleosides that may include a phosphonate moiety astherapeutic molecules for treatment of hepatitis B, C, and D, orproliferative disorders. However, they report only a therapeutic effectagainst HBV. In still further known examples, (see e.g., U.S. Pat. Nos.5,142,051, 5,302,585, 5,208,221, or 5,356,886) various acyclicphosphonate nucleoside analogs are presented with variouspharmacological effects. However, such compounds are frequentlydifficult to synthesize and/or exhibit less than desirable (if any)antiviral activity against HCV.

[0008] In yet further examples, inorganic phosphonates (e.g., Foscarnet)have been employed to treat HBV infection as described in U.S. Pat. No.6,495,521 to Horwitz, while various pyrophosphate analogs were shown toexhibit antiviral effect as described by McKenna et al in U.S. Pat. No.6,444,837. However, despite relatively promising antiviral activity ofsuch compounds against certain viruses, inorganic phosphonates andpyrophosphate analogs appear to exhibit less than desirable (if any)antiviral activity against HCV.

[0009] Therefore, although various compounds and methods are known inthe art to treat HCV infection, all or almost all of them suffer fromone or more disadvantages. Consequently, there is still a need forimproved compounds and methods to treat viral infections, andparticularly HCV infections.

SUMMARY OF THE INVENTION

[0010] The present invention is generally directed to compositions andmethods of treatment of HCV infections, and particularly relates tonucleotide analogs having a phosphonate moiety and pharmaceuticalcompositions comprising the same.

[0011] In one especially preferred aspect, contemplated compositionsinclude a nucleotide analog having a structure according to Formula 1 or2 (substituents defined as in the section “Contemplated Compounds”below), wherein the nucleotide analog is present in the compositioneffective to inhibit a viral polymerase of an HCV virus and/or to act asa substrate for the viral polymerase of the HCV virus.

[0012] Further preferred nucleotide analogs particularly include thosein which X is a covalent bond between the C4′-atom of the sugar and thecarbon atom in the phosphonate group, O, or CH₂, and wherein at leastone of R_(1′, R) ₂′, R₃′, and R₄′ is CH₃. With respect to heterocyclicbases, it is preferred that Z₁ and Z₂ is H, and W is NR⁵R⁶, and that Yis H or CH₃, and V is OH or NR⁵R⁶. It is still further contemplated thatthe viral polymerase is disposed in a cell infected with HCV, and mostpreferably that the cell is disposed in a patient infected with HCV.

[0013] In another aspect of the inventive subject matter, prodrugs ofthe nucleotide analog are contemplated, and particularly preferredprodrugs include a moiety that is preferentially removed from theprodrug in a hepatocyte. Exemplary preferred moieties are covalentlybound to the phosphonate group and comprise an amino acid, or form acyclic group with the phosphonate group. Alternatively, or additionally,pharmaceutically acceptable salts of the nucleotide analogs according tothe inventive subject matter are contemplated.

[0014] Various objects, features, aspects and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention and theaccompanying drawing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 depicts one contemplated route for the synthesis ofexemplary 5′-deoxy-5′-phosphonate nucleosides.

[0016]FIG. 1A depicts another contemplated route for the synthesis ofexemplary 5′-deoxy-5′-phosphonate nucleosides.

[0017]FIG. 2 depicts a further contemplated route for the synthesis ofexemplary 5′-deoxy-5′-phosphonate nucleosides.

[0018]FIG. 3 depicts one contemplated route for the synthesis ofexemplary 5′-deoxy-5′-methylenephosphonate nucleosides.

[0019]FIG. 3A depicts another contemplated route for the synthesis ofexemplary 5′-deoxy-5′-methylenephosphonate nucleosides.

[0020]FIG. 3B depicts a further contemplated route for the synthesis ofexemplary 5′-deoxy-5′-methylenephosphonate nucleosides.

[0021]FIG. 4 depicts yet another contemplated route for the synthesis ofexemplary 5′-deoxy-5′-methylenephosphonate nucleosides.

[0022]FIG. 4A depicts a still further contemplated route for thesynthesis of exemplary 5′-deoxy-5′-methylenephosphonate nucleosides.

[0023]FIG. 5 depicts one contemplated route for the synthesis ofexemplary 5′-phosphonate nucleotide analogs.

[0024]FIG. 6 depicts another contemplated route for the synthesis ofexemplary sugar-modified 5′-phosphonate nucleotide analogs.

[0025]FIG. 7 depicts yet another contemplated route for the synthesis ofexemplary 5′-phosphonate nucleotide analogs.

DETAILED DESCRIPTION

[0026] The term “nucleoside” as used herein refers to all compounds inwhich a heterocyclic base is covalently coupled to a sugar. The term“nucleoside analog” as used herein refers to a nucleoside in which atleast one of the sugar and the heterocyclic base is a non-natuarallyoccurring sugar and/or heterocyclic base. An especially preferredcoupling of the base to the sugar is a C1′-(glycosidic) bond, in whichthe C1′ carbon atom of the sugar is covalently linked to a carbon- orheteroatom (typically nitrogen) in the base. The term “nucleotide” asused herein refers to a nucleoside that is coupled to a phosphate group(or modified phosphate group, including phosphonate,hytdroxymethylphosphonate, etc.), preferably via the C4′- or C5′-atom.The terms “nucleoside”, “nucleotide”, “nucleoside analog”, and“nucleotide analog” are also employed broadly herein to include allprodrug compositions that are activated or converted in a human to anucleoside or nucleotide (or analog thereof) in one or more than onestep, which step(s) may occur intracellularly or extracellularly.Especially contemplated prodrug forms include those that confer aparticular specificity towards a diseased or infected cell or organ.Exemplary contemplated prodrug forms are described in “Prodrugs” byKenneth B. Sloan (Marcel Dekker; ISBN: 0824786297), “Design of Prodrugs”by Hans Bundgaard (ASIN: 044480675X), and in copending U.S. applicationSer. No. 09/594410, filed Jun. 16, 2000. Further preferred prodrugs arefound in the section entitled “Contemplated Compounds” below.

[0027] The term “alkyl” as used herein refers to any linear, branched,or cyclic, primary, secondary, or tertiary hydrocarbon in which allcarbon-carbon bonds are single bonds. This term specifically includesmethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl,tertiobutyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl,cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. The term “alkyl” also specifically includes bothsubstituted and unsubstituted alkyls, e.g., where a substituent is afunctional group. Particularly contemplated functional groups includenucleophilic (e.g., —NH₂, —OH, —SH, —NC, etc.) and electrophilic groups(e.g., C(O)OR, C(X)OH, etc.), polar groups (e.g., —OH), ionic groups(e.g., —NH₃ ⁺), and halogens (e.g., —F, —Cl), and all chemicallyreasonable combinations thereof.

[0028] The term “lower alkyl” is used herein and, unless otherwisespecified, refers to a C₁ to C₄ saturated linear, branched, or cyclic(for example cyclopropyl), alkyl group, including both substituted andunsubstituted forms. Unless otherwise specifically stated in thisapplication, when alkyl is a suitable moiety, lower alkyl is preferred.Similarly, when alkyl or lower alkyl is a suitable moiety, unsubstitutedalkyl or lower alkyl is preferred.

[0029] The terms “alkenyl” and “unsubstituted alkenyl” are usedinterchangeably herein and refer to any linear, branched, or cyclicalkyl with at least one carbon-carbon double bond. The term “substitutedalkenyl” as used herein refers to any alkenyl that further comprises afunctional group, and particularly contemplated functional groupsinclude those discussed above. The terms “alkynyl” and “unsubstitutedalkynyl” are used interchangeably herein and refer to any linear,branched, or cyclic alkyl or alkenyl with at least one carbon-carbontriple bond. The term “substituted alkynyl” as used herein refers to anyalkynyl that further comprises a functional group, and particularlycontemplated functional groups include those discussed above.

[0030] The terms “aryl” and “unsubstituted aryl” are usedinterchangeably herein and refer to any aromatic cyclic alkenyl oralkynyl. The term “substituted aryl” as used herein refers to any arylthat further comprises a functional group, and particularly contemplatedfunctional groups include those discussed above. The term “alkaryl” isemployed where the aryl is further covalently bound to an alkyl,alkenyl, or alkynyl.

[0031] The term “protected” as used herein and unless otherwise definedrefers to a group that is covalently coupled to an oxygen, nitrogen, orphosphorus atom to prevent further reaction or for other purposes. Awide variety of oxygen, nitrogen, and phosphorus protecting groups areknown to those skilled in the art of organic synthesis.

[0032] The terms “purine”, “deazapurine”, “pyrimidine”, “imidazole” or“triazole base” include adenine, guanine, hypoxanthine,2,6-diaminopurine, 6-chloropurine, N⁶-alkylpurines, N⁶-acylpurine(wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl),N⁶-benzylpurine, N⁶-halopurine, N⁶-vinylpurine, N⁶-acetylenic purine,N⁶-hydroxyalkyl purine, N⁶-thioalkyl purine, N²-alkyl purines,N²-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine,5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or4-mercaptopyrimidine, uracil, 5-halouracil, including 5-fluorouracil,C⁵-alkylpyrimidine, C⁵-benzylpyrimidine, C⁵-halopyrimidine,C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine, C⁵-acylpyrimidine,C⁵-hydroxyalkyl pyrimidine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine,C⁵-nitropyrimidine, C⁵-aminopyrimidine, 5-azacytosine, 5-azauracil,triazolopyrimidine, imidazolopyrimidine, pyrrolopyrimidine,pyrazolopyrimidine, triazolopyridine, imidazolopyridine, pyrrolopyridineand pyrazolopyridine, C⁴-carbamoyl imidazole, C³-cyano-1,2,4-triazole,C³-carbamoyl-1,2,4-triazole, C³-carboxamidine-1,2,4-triazole. Functionaloxygen and nitrogen groups on the base can be protected as necessary ordesired. Suitable protecting groups are well known to those skilled inthe art, and include trimethylsilyl, dimethylhexylsilyl,tert-butyldimethylsilyl, and tert-butyldiphenylsilyl, trityl, alkylgroups, and acyl groups such as acetyl and propionyl, methanesulfonyl,and para-toluenesulfonyl.

[0033] Still further, the terms “halo” and “halogen” are usedinterchangeably herein and include chloro, bromo, iodo, and fluoro.

[0034] The term “substantially free of” or “substantially in the absenceof” refers to a nucleoside phosphonate composition that includes atleast 85% or 90% by weight, preferably 95% to 98% by weight, and evenmore preferably 99% to 100% by weight, of the designated isomer (e.g.,D-isomer or L-isomer) of that nucleoside phosphonate. In a preferredaspect of the inventive subject matter, in the methods and compounds ofthis invention, the compounds are D-isomers and substantially free ofthe corresponding L-isomer.

Contemplated Compounds

[0035] The inventors discovered that various nucleoside analogs, theirsalts and prodrugs may be employed as inhibitors and/or substrates for anon-mammalian polymerase, and especially as inhibitors of viral andbacterial polymerases, wherein particularly contemplated viralpolymerases include RNA-dependent RNA polymerases (which may be de novopolymerases), and most particularly the HCV polymerase NS5B.

[0036] Furthermore, the inventors contemplate that contemplatedphosphonates and their prodrugs may enter a cell and will be—afterintracellular phosphorylation—converted to their diphosphatederivatives. Therefore, it is especially contemplated that nucleosidephosphonate diphosphates (nucleoside triphosphate mimics) may beemployed by an RNA-dependent RNA polymerase (and preferably the HCVpolymerase) as a nucleotide for incorporation into viral RNA.

[0037] In one particularly preferred aspect, contemplated nucleotidesand nucleotide analogs will have a structure according to the generalformula:

P—X—S-Base

[0038] wherein P includes a phosphonate (or modified phosphonate,including a phosphoamidate group) that is coupled to a sugar S via abond, atom, or group X. Further coupled to the sugar S is a heterocyclicbase “Base”. Thus, contemplated compounds preferbaly comprise anucleotide in which a phosphonate group or modified phosphonate group iscovalently coupled to the sugar of the nucleotide via a group other thana phosphate ester, and wherein the nucleotide is at least one of asubstrate and an inhibitor of a viral polymerase.

[0039] Preferred phosphonate groups include those that are modified witha cleavable moiety, wherein cleavage is preferably achievedintracellularly by one or more enzymes. Consequently, particularlypreferred modified phosphonate groups include moieties covalentlycoupled to the phosphonate as those described in U.S. Pat. No. 6,312,662to Erion, which is incorporated by reference herein.

[0040] Preferred sugars have the general formula of C_(n)H_(2n)O_(n),wherein n is between 2 and 8, and where applicable, the sugar is in theD- or L-configuration. Moreover, it should be appreciated that there arenumerous equivalent modifications of such sugars known in the art (sugaranalogs), and all such modifications are specifically included herein.For example, some contemplated alternative sugars will include sugars inwhich the heteroatom in the cyclic portion of the sugar is an atom otherthan oxygen (e.g., sulfur, carbon, or nitrogen) analogs, while otheralternative sugars may not be cyclic but in a linear (open-chain) form.Suitable sugars may also include one or more double bonds. Still furtherspecifically contemplated alternative sugars include those with one ormore non-hydroxyl substituents, and particularly contemplatedsubstituents include mono-, di-, and triphosphates (preferably as C₅′esters), alkyl groups, alkoxy groups, halogens, amino groups and amines,sulfur-containing substituents, etc.

[0041] Particularly contemplated modifications include substitutedribofuranoses, wherein the substituent is a substituent (preferably inbeta orientation) on the 2′- and/or 3′-carbon atoms, most preferably analkyl, alkenyl, alkynyl, aryl, heterocycle, CF₃, CF₂H, CCl₃, CCl₂H,CH₂OH, CN, COOR′, and CONHR′, with R′ being C₁₋₁₀ alkyl, alkenyl,alkynyl, aryl. However, it should be appreciated that all contemplatedsubstituents (hydroxyl substituents and non-hydroxyl substituents) maybe directed in the alpha or the beta position.

[0042] Numerous contemplated sugars and sugar analogs are commerciallyavailable. Where contemplated sugars are not commercially available, itshould be recognized that there are various methods known in the art tosynthesize such sugars. For example, suitable protocols can be found in“Modern Methods in Carbohydrate Synthesis” by Shaheer H. Khan (Gordon &Breach Science Pub; ISBN: 3718659212), in U.S. Pat Nos. 4,880,782, and3,817,982, and in WO88/00050, or in EP199,451.

[0043] Similarly, contemplated heterocyclic bases preferably comprise aheterocyclic base that can form at least one hydrogen bond to anotherheterocyclic base in a (oligo- or poly-) nucleoside or (oligo- or poly-)nucleotide. Consequently, contemplated heterocyclic bases includecompounds in which a plurality of atoms (wherein at least one atom is anatom other than a carbon atom) form a ring via a plurality of covalentbonds. However, particularly contemplated heterocyclic bases havebetween one and three rings, wherein especially preferred rings include5- and 6-membered rings with nitrogen, sulfur, or oxygen as the non-carbon atom (e.g., imidazole, pyrrole, triazole, dihydropyrimidine).Further contemplated heterocycles may be fused (i.e., covalently bound)to another ring or heterocycle, and are thus termed “fused heterocycles”as used herein. Especially contemplated fused heterocycles include a5-membered ring fused to a 6-membered ring (e.g., purine,pyrrolo[2,3-d]pyrimidine), and a 6-membered ring fused to another6-membered or higher ring (e.g., pyrido[4,5-d]pyrimidine,benzodiazepine).

[0044] Preferred groups X of the general formula provided above willinclude all atoms/groups that replace the C5′-carbon of the sugar in thenucleotide (analog) with a methylene group (which may or may not besubstituted, and preferably halogenated), an oxygen atom, a sulfur atom,or a covent bond (thus forming a bond between the C4′-atom of the sugarand the carbon atom in the phosphonate group).

[0045] In one preferred aspect, compounds of Formulae (I)-(IV) orpharmaceutically acceptable salts or prodrugs thereof are contemplated

[0046] wherein R¹ and R² are independently H, phosphate, diphosphate, ora group that is preferentially removed in a hepatocyte to yield thecorresponding OH group. The term “preferentially removed in ahepatocyte” as used herein means at least part of the group is removedin a hepatocyte at a rate higher than the rate of removal of the samegroup in a non-hepatocytic cell (e.g., fibroblast or lymphocyte). It istherefore contemplated that the removable group includes allpharmaceutically acceptable groups that can be removed by a reductase,esterase, cytochrome P450 or any other specific liver enzyme.Alternative contemplated groups may also include groups that are notnecessarily preferentially removed in a hepatocyte, but effect at leastsome accumulation and/or specific delivery to a hepatocyte (e.g., esterswith selected amino acids, including valine, leucine, isoleucine, orpolyarginine or polyaspartate).

[0047] R³ and R⁴ are independently H, phosphate (including mono-, di- ortriphosphate), acyl (especially including lower acyl), alkyl (especiallyincluding lower alkyl), sulfonate ester, including alkyl or arylalkylsulfonyl including methane sulfonyl and benzyl (wherein the phenyl groupis optionally substituted with one or more substituents) lipids,including phospholipids, amino acids, carbohydrates, peptides,cholesterol, or another pharmaceutically acceptable leaving group which,when administered in vivo is capable of providing a compound wherein R³and R⁴ is H.

[0048] W is hydrogen, bromo, chloro, fluoro, iodo, OR⁵, NR⁵R⁶,NH(NR⁵R⁶), N(alkyl)(NR⁵R⁶), CN, C(O)NR⁵R⁶, C(NH)NR⁵R⁶ or SR⁵; Z¹ and Z²are independently selected from the group consisting of H, alkyl,CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OR⁵,NR⁶R⁷ or SR⁵; and R⁵ and R⁶ are independently H, acyl (especiallyincluding lower acyl), or alkyl (and especially including methyl, ethyl,propyl and cyclopropyl). In particularly preferred aspects of Formulae(I)-(IV), the compound is in the form of a prodrug or a pharmaceuticallyacceptable salt thereof, and additionally or alternatively, Z₁ and Z₂ incontemplated compounds is H, while W is NR⁵R⁶.

[0049] In another preferred aspect, compounds of Formulae (V)-(VIII) orpharmaceutically acceptable salts or prodrugs thereof are contemplated

[0050] wherein R¹ and R² are independently H, phosphate, diphosphate, ora group that is preferentially removed in a hepatocyte to yield thecorresponding OH group. Thus, contemplated groups include allpharmaceutically acceptable groups that can be removed by a reductase,esterase, cytochrome P450 or any other specific liver enzyme.Alternative contemplated groups may also include groups that are notnecessarily preferentially removed in a hepatocyte, but effect at leastsome accumulation and/or specific delivery to a hepatocyte (e.g., esterswith selected amino acids, including valine, leucine, isoleucine, orpolyarginine or polyaspartate).

[0051] R³ and R⁴ are independently H, phosphate (including mono-, di- ortriphosphate), acyl (especially including lower acyl), alkyl (especiallyincluding lower alkyl), sulfonate ester, including alkyl or arylalkylsulfonyl including methane sulfonyl and benzyl (wherein the phenyl groupis optionally substituted with one or more substituents) lipids,including phospholipids, amino acids, carbohydrates, peptides,cholesterol, or another pharmaceutically acceptable leaving group which,when administered in vivo is capable of providing a compound wherein R³and R⁴ is H.

[0052] V is hydrogen, bromo, chloro, fluoro, iodo, OR⁵, NR⁵R⁶,NH(NR⁵R⁶), N(alkyl)(NR⁵R⁶), CN, C(O)NR⁵R⁶, C(NH)NR⁵R⁶ or SR⁵; Y is H,alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo, fluoro, iodo,OR⁵, NR⁶R⁷, or SR⁵; and R⁵ and R⁶ are independently H, acyl (especiallyincluding lower acyl), or alkyl (and especially including methyl, ethyl,propyl and cyclopropyl). In particularly preferred aspects of Formulae(V)-(VIII), the compound is in the form of a prodrug or apharmaceutically acceptable salt thereof, and additionally oralternatively, Y in contemplated compounds is H or CH₃, while V is OH orNR⁵R⁶.

[0053] Of course, it should be recognized that the compounds of Formulae(I)-(IV) may be prepared such that the C5′-atom of the sugar is replacedby a heteroatom, and most preferably an oxygen atom. Thus, in a furtherpreferred aspect, contemplated compounds may have a structure accordingto Formulae (IX)-(XII), wherein the substituents R¹, R², R³, R⁴, V and Yare defined as described for compounds according to Formulae (V)-(VIII)above:

[0054] Similarly, corresponding modifications (i.e., replacement ofC5′-atom with oxygen) may be made for the compounds according toFormulae (V)-(VIII) to provide the compounds according to Formulae(XIII)-(XVI) wherein the substituents R¹, R², R³, R⁴, Z¹, Z², and W aredefined as described for compounds according to Formulae (I)-(IV) above:

[0055] In a still further preferred aspect of the inventive subjectmatter, contemplated nucleotide analogs may be prepared such that thephosphonate moiety is directly and covalently bound to the C5′-atom ofthe sugar, or via an alkylene (or other) linker as depicted in thecompounds according to Formulae (XVII)-(XIX). Alternatively, oradditionally, such compounds may include modified sugar moieties asdepicted below.

[0056] Here, the heterocyclic bases of the nucleotide analogs includeall suitable heterocyclic bases (e.g., substituted purine, deaza purine,pyrimidine, imidazole, or triazole). However, particularly preferredheterocyclic bases especially include those as described in compoundsaccording to Formulae (I) and (V). Consequently, particularly preferredcompound will have a general structure according to Formulae(XVII)-(XIX), and may be present in the form of a pharmaceuticallyacceptable salt or prodrug thereof:

[0057] in which n is 0, 1, 2 or 3 (alternatively, one or more CH₂ can bereplaced by CHF, CF₂, CHCl, CCl₂, CHBr, CBr₂, CH(CN), or CH(alkyl)); X¹and X² are independently O, S or NH; Q is O or S when n=1, 2 or 3; Q isCH₂ when n=0;

[0058] R¹ and R² are independently H, phosphate, diphosphate, or a groupthat is preferentially removed in a hepatocyte to yield thecorresponding OH group. Thus, contemplated groups include allpharmaceutically acceptable groups that can be removed by a reductase,esterase, cytochrome P450 or any other specific liver enzyme.Alternative contemplated groups may also include groups that are notnecessarily preferentially removed in a hepatocyte, but effect at leastsome accumulation and/or specific delivery to a hepatocyte (e.g., esterswith selected amino acids, including valine, leucine, isoleucine, orpolyarginine or polyaspartate).

[0059] R³ and R⁴ are independently H, phosphate (including mono-, di- ortriphosphate), acyl (especially including lower acyl), alkyl (especiallyincluding lower alkyl), sulfonate ester, including alkyl or arylalkylsulfonyl including methane sulfonyl and benzyl (wherein the phenyl groupis optionally substituted with one or more substituents) lipids,including phospholipids, amino acids, carbohydrates, peptides,cholesterol, or another pharmaceutically acceptable leaving group which,when administered in vivo is capable of providing a compound wherein R³and R⁴ is H.

[0060] R⁵ is hydrogen, hydroxy, alkyl (including lower alkyl), alkoxy,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —S(alkyl), —S(O)(alkyl), —S(O)(O)(alkyl), —O(alkenyl), CF3,CHF2, CH2F, CH2OH, CH2O(lower alkyl), chloro, bromo, fluoro, iodo, NO₂,NH₂, —NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;

[0061] Y is O, S, SO₂, C(═CH₂), NR or CH₂; R is H, acyl (including loweracyl) or alkyl (including lower alkyl).

[0062] In a first preferred aspect of Formulae (XVII)-(XIX), a compoundis provided wherein n is 1, Q is O, and the Base is as described incompounds according to Formulae (I) and (V). R¹ and R² are independentlyH, phosphate (including monophosphate, diphosphate and stabilizedphosphate prodrugs) or any pharmaceutically acceptable group (includinggroups activated by reductase, esterase, cytochrome or any other liverenzyme) known to undergo a chemical modification resulting in therelease of the compound wherein R¹ and R² is an H; R³ and R⁴ are H; R⁵is alkyl; X¹ and X² are O; and Y is O, S or CH₂.

[0063] In a second preferred aspect of Formulae (XVII)-(XIX), a compoundis provided wherein n is 1, Q is CH₂, and the Base is as described incompounds according to Formulae (I) and (V). R¹ and R² are independentlyH, phosphate (including monophosphate, diphosphate and stabilizedphosphate prodrugs) or any pharmaceutically acceptable group (includinggroups activated by reductase, esterase, cytochrome or any other liverenzyme) known to undergo a chemical modification resulting in therelease of the compound wherein R¹ and R² is an H; R³ and R⁴ are H; R⁵is alkyl; X¹ and X² are O; and Y is O, S or CH₂.

[0064] In a third preferred aspect of Formulae (XVII)-(XIX), a compoundis provided wherein n is 1, Q is O or CH₂, and the Base is as describedin compounds according to Formulae (I) and (V). R¹ and R² areindependently H, phosphate (including monophosphate, diphosphate andstabilized phosphate prodrugs) or any pharmaceutically acceptable group(including groups activated by reductase, esterase, cytochrome or anyother liver enzyme) known to undergo a chemical modification resultingin the release of the compound wherein R¹ and R² is an H; R³ and R⁴ areH; R⁵ is alkyl; X¹ and X² are O; and Y is O.

[0065] Similarly, the R⁵ substituent (and especially where R5 is asubstituent other than H) may also be in a position other than inbeta-orientation at the C1′-atom, and where it is preferred that thealternative position is at the C2′-atom, compounds according to Formula(XX)-(XXII) are especially contemplated. With respect to thesubstituents R¹, R², X¹, X², Q, Y, R³, R⁴, and R₅ and the number n, thesame considerations as described above for compounds according toFormulae (XVII)-(XIX) apply.

[0066] In a first preferred aspect of Formulae (XX)-(XXII), n is 1, Q isO, and the Base is as described in compounds according to Formulae (I)and (V). R¹ and R² are independently H, phosphate (includingmonophosphate, diphosphate and stabilized phosphate prodrugs) or anypharmaceutically acceptable group (including groups activated byreductase, esterase, cytochrome or any other liver enzyme) known toundergo a chemical modification resulting in the release of the compoundwherein R¹ and R² is an H; R³ and R⁴ are H; R⁵ is alkyl; X¹ and X² areO; and Y is O, S or CH₂.

[0067] In a second preferred aspect of Formulae (XX)-(XXII), a compoundis provided wherein n is 1, Q is CH₂, and the Base is as described incompounds according to Formulae (I) and (V). R¹ and R² are independentlyH, phosphate (including monophosphate, diphosphate and stabilizedphosphate prodrugs) or any pharmaceutically acceptable group (includinggroups activated by reductase, esterase, cytochrome or any other liverenzyme) known to undergo a chemical modification resulting in therelease of the compound wherein R¹ and R² is an H; R³ and R⁴ are H; R⁵is alkyl; X¹ and X² are O; and Y is O, S or CH₂.

[0068] In a third preferred aspect, of Formulae (XX)-(XXII), a compoundis provided wherein n is 1, Q is O or CH₂, and the Base is as describedin compounds according to Formulae (I) and (V). R¹ and R² areindependently H, phosphate (including monophosphate, diphosphate andstabilized phosphate prodrugs) or any pharmaceutically acceptable group(including groups activated by reductase, esterase, cytochrome or anyother liver enzyme) known to undergo a chemical modification resultingin the release of the compound wherein R¹ and R² is an H; R³ and R⁴ areH; R⁵ is alkyl; X¹ and X² are O; and Y is O.

[0069] Furthermore, the R⁵ substituent (and especially where R⁵ is asubstituent other than H) may also be in a position other than inbeta-orientation at the C1′- or C2′-atom, and where it is preferred thatthe alternative position is at the C3′-atom, compounds according toFormula (XXIII)-(XXV) are especially contemplated. With respect to thesubstituents R¹, R², X¹, X², Q, Y, R³, R⁴, and R⁵ and the number n, thesame considerations as described above for compounds according toFormulae (XVII)-(XIX) apply.

[0070] In a first preferred aspect of Formulae (XXIII)-(XXV), a compoundis provided wherein n is 1, Q is O, and the Base is as described incompounds according to Formulae (I) and (V). R¹ and R² are independentlyH, phosphate (including monophosphate, diphosphate and stabilizedphosphate prodrugs) or any pharmaceutically acceptable group (includinggroups activated by reductase, esterase, cytochrome or any other liverenzyme) known to undergo a chemical modification resulting in therelease of the compound wherein R¹ and R² is an H; R³ and R⁴ are H; R⁵is alkyl; X¹ and X² are O; and Y is O, S or CH₂.

[0071] In a second preferred aspect of Formulae (XXIII)-(XXV), acompound is provided wherein n is 1, Q is CH₂, and the Base is asdescribed in compounds according to Formulae (I) and (V). R¹ and R² areindependently H, phosphate (including monophosphate, diphosphate andstabilized phosphate prodrugs) or any pharmaceutically acceptable group(including groups activated by reductase, esterase, cytochrome or anyother liver enzyme) known to undergo a chemical modification resultingin the release of the compound wherein R¹ and R² is an H; R³ and R⁴ areH; R⁵ is alkyl; X¹ and X² are O; and Y is O, S or CH₂.

[0072] In a third preferred aspect, of Formulae (XXIII)-(XXV), acompound is provided wherein n is 1, Q is O or CH₂, and the Base is asdescribed in compounds according to Formulae (I) and (V). R¹ and R² areindependently H, phosphate (including monophosphate, diphosphate andstabilized phosphate prodrugs) or any pharmaceutically acceptable group(including groups activated by reductase, esterase, cytochrome or anyother liver enzyme) known to undergo a chemical modification resultingin the release of the compound wherein R¹ and R² is an H; R³ and R⁴ areH; R⁵ is alkyl; X¹ and X² are O; and Y is O.

[0073] Still further, the R⁵ substituent (and especially where R5 is asubstituent other than H) may also be the C4′-atom as depicted incompounds according to Formula (XXVI)-(XXVIII). Again, with respect tothe substituents R¹, R², X¹, X², Q, Y, R³, R⁴, and R₅ and the number n,the same considerations as described above for compounds according toFormulae (XVII)-(XIX) apply.

[0074] In a first preferred aspect of Formulae (XXVI)-(XXVIII), acompound is provided wherein n is 1, Q is O, and the Base is asdescribed in compounds according to Formulae (I) and (V). R¹ and R² areindependently H, phosphate (including monophosphate, diphosphate andstabilized phosphate prodrugs) or any pharmaceutically acceptable group(including groups activated by reductase, esterase, cytochrome or anyother liver enzyme) known to undergo a chemical modification resultingin the release of the compound wherein R¹ and R² is an H; R³ and R⁴ areH; R⁵ is alkyl; X¹ and X² are O; and Y is O, S or CH₂.

[0075] In a second preferred aspect of Formulae (XXVI)-(XXVIII), acompound is provided wherein n is 1, Q is CH₂, and the Base is asdescribed in compounds according to Formulae (I) and (V). R¹ and R² areindependently H, phosphate (including monophosphate, diphosphate andstabilized phosphate prodrugs) or any pharmaceutically acceptable group(including groups activated by reductase, esterase, cytochrome or anyother liver enzyme) known to undergo a chemical modification resultingin the release of the compound wherein R¹ and R² is an H; R³ and R⁴ areH; R⁵ is alkyl; X¹ and X² are O; and Y is O, S or CH₂.

[0076] In a third preferred aspect of Formulae (XXVI)-(XXVIII), acompound is provided wherein n is 1, Q is O or CH₂, and the Base is asdescribed in compounds according to Formulae (I) and (V). R¹ and R² areindependently H, phosphate (including monophosphate, diphosphate andstabilized phosphate prodrugs) or any pharmaceutically acceptable group(including groups activated by reductase, esterase, cytochrome or anyother liver enzyme) known to undergo a chemical modification resultingin the release of the compound wherein R¹ and R² is an H; R³ and R⁴ areH; R⁵ is alkyl; X¹ and X² are O; and Y is O.

[0077] Finally, the R⁵ substituent (and especially where R5 is asubstituent other than H) may also be bound to the C5′-atom to provide a“branched” sugar as depicted in compounds according to Formulae(XXIX)-(XXXI). Once more, with respect to the substituents R¹, R², X¹,X², Q, Y, R³, R⁴, and R⁵ and the number n, the same considerations asdescribed above for compounds according to Formulae (XVII)-(XIX) apply.

[0078] In a first preferred aspect of Formulae (XXIX)-(XXXI), a compoundis provided wherein n is 0, and the Base is as described in compoundsaccording to Formulae (I) and (V). R¹ and R² are independently H,phosphate (including monophosphate, diphosphate and stabilized phosphateprodrugs) or any pharmaceutically acceptable group (including groupsactivated by reductase, esterase, cytochrome or any other liver enzyme)known to undergo a chemical modification resulting in the release of thecompound wherein R¹ and R² is an H; R³ and R⁴ are H; R⁵ is H; X¹ and X²are O; and Y is O, S or CH₂.

[0079] In a second preferred aspect of Formulae (XXIX)-(XXXI), acompound is provided wherein n is 1, and the Base is as described incompounds according to Formulae (I) and (V). R¹ and R² are independentlyH, phosphate (including monophosphate, diphosphate and stabilizedphosphate prodrugs) or any pharmaceutically acceptable group (includinggroups activated by reductase, esterase, cytochrome or any other liverenzyme) known to undergo a chemical modification resulting in therelease of the compound wherein R¹ and R² is an H; R³ and R⁴ are H; R⁵is H; X¹ and X² are O; and Y is O, S or CH₂.

[0080] In a third preferred aspect of Formulae (XXIX)-(XXXI), a compoundis provided wherein n is 1, and the Base is as described in compoundsaccording to Formulae (I) and (V). R¹ and R² are independently H,phosphate (including monophosphate, diphosphate and stabilized phosphateprodrugs) or any pharmaceutically acceptable group (including groupsactivated by reductase, esterase, cytochrome or any other liver enzyme)known to undergo a chemical modification resulting in the release of thecompound wherein R¹ and R² is an H; R³ and R⁴ are H; R⁵ is alkyl; X¹ andX² are O; and Y is O.

[0081] In a still further particularly preferred aspect, compoundsaccording to Formulae (XXXII)-(XXXIII), or a pharmaceutically acceptablesalt or prodrug thereof, are provided:

[0082] wherein n is 0, 1, 2 or 3. Alternatively, one or more CH₂ can bereplaced by CHF, CF₂, CHCl, CCl₂, CHBr, CBr₂, CH(CN), or CH(alkyl), andthe Base is as described in compounds according to Formulae (I) and (V).

[0083] R¹ and R² are independently H, phosphate (includingmonophosphate, diphosphate and stabilized phosphate prodrugs) or anypharmaceutically acceptable group (including groups activated byreductase, esterase, cytochrome or any other liver enzyme) known toundergo a chemical modification resulting in the release of the compoundwherein R¹ and R² is an H;

[0084] R⁵, R⁹, R¹⁰, R¹¹ and R¹² are independently selected fromhydrogen, hydroxy, alkyl (including lower alkyl), alkoxy, azido, cyano,alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl), —C(O)O(loweralkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(lower alkyl), —S(alkyl),—S(O)(alkyl), —S(O)(O)(alkyl), —O(alkenyl), CF3, CHF2, CH2F, CH2OH,CH2O(lower alkyl), chloro, bromo, fluoro, iodo, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;

[0085] R⁷ and R⁸ are independently hydrogen, OR³, hydroxy, alkyl(including lower alkyl), alkoxy, azido, cyano, alkenyl, alkynyl,Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl), —C(O)O(lower alkyl), —O(acyl),—O(lower acyl), —O(alkyl), —O(lower alkyl), —S(alkyl), —S(O)(alkyl),—S(O)(O)(alkyl), —O(alkenyl), CF3, CHF2, CH2F, CH2OH, CH2O(lower alkyl),chloro, bromo, fluoro, iodo, NO₂, NH₂, —NH(lower alkyl), —NH(acyl),—N(lower alkyl)₂, —N(acyl)₂; alternatively, R⁷ and R⁹, R⁷ and R¹⁰, R⁸and R⁹, or R⁸ and R¹⁰ can come together to form pi bonds;

[0086] Q is O or S when n=1, 2 or 3; Q is CH₂ when n=0; X¹ and X² areindependently O, S or NH; Y is O, S, SO₂, C(═CH₂), NR⁶ or CH₂; and R⁶ isH, acyl (including lower acyl), or alkyl (including lower alkyl).

[0087] In yet further contemplated aspects of the inventive subjectmatter, it is contemplated that the carbon atom of the phosphonate groupmay be substituted with hydrogen atoms, halogen atoms, and one hydrogenatom and one halogen atom. Thus, contemplated compounds also includehalophosphonate nucleotide analogs and a mixed halophosphonatenucleotide analog (i.e., with one hydrogen atom and one halogen atom atthe carbon of the phosphonate group).

[0088] Moreover, it should be recognized that all prodrugs andmetabolites of the compounds according to Formulae (I)-(XXIII) arecontemplated. There are numerous prodrug modifications ofpharmacologically active molecules known in the art, and all of suchmodifications are considered suitable for use herein. However,especially preferred prodrugs include those that deliver contemplatedcompounds to a target cell (e.g., hepatocyte infected with HCV) ortarget organ (e.g., liver infected with HCV), wherein the prodrug formmay be converted within a cell, organ, or other body compartment in anenzymatic or non-enzymatic manner. Especially preferred prodrug formsinclude those described in WO 99/45016 and various esters withpreferably lipophilic amino acids. Further preferred prodrugsparticularly include those in which the prodrug form is less active ascompared to the corresponding non-prodrug form. Thus, specificallypreferred compounds may include a moiety that increases uptake of theprodrug into a cell, or that increases preferential retention of thecompound (which may or may not be in prodrug form) in a cell.Alternatively, contemplated compounds may be formulated to increasetarget specificity of the compound (e.g., organ specific liposomes).

[0089] With respect to the metabolite, it should be recognized thatmetabolites of contemplated compounds might be formed by one or moreenzymatic reactions (e.g., via hydrolysis, oxidation, reduction, lyase,or ligase reaction, or even via a polymerase action), or vianon-enzymatic reactions (e.g., acid hydrolysis, reduction). For example,a hydrolase or lyase may cleave a portion of contemplated compounds to amore active form. On the other hand, reactions of hydroxylases, ligases,or other enzymes that add chemical groups to the compounds according tothe inventive subject matter (to render the compounds more active) arealso contemplated herein. Thus, it should be recognized that allmetabolites that have a desirable therapeutic effect, and especially anantiviral effect, are deemed suitable.

Contemplated Activity and Uses

[0090] The inventors surprisingly discovered that the compoundsaccording to the inventive subject matter inhibit non-mammalianpolymerases and/or act as a substrate for non-mammalian polymerases.Particularly contemplated polymerases include viral and bacterialpolymerases, and most preferably viral RNA-dependent RNA polymerases(e.g., HCV polymerase NS5B). The term “act as a substrate for thepolymerase” as used herein means that the compound is incorporated via acovalent bond into a nascent oligo-or polynucleotide that is synthesizedby the polymerase, wherein the compound may then either act as a chainterminator or as a non-chain-terminator (i.e., the compound will formpart of a polynucleotide in which the compound is not at the 3′-end ofthe polynucleotide). Of course it should be recognized that wherecontemplated compounds act as a substrate, such compounds may be presentin diphosphate or triphosphate form (either synthetic, or phosphorylatedin vitro or in vivo by a kinase).

[0091] Still further, it should be recognized that inhibition of thepolymerase may be specific, relative to a mammalian polymerse, andsuitable specificities are generally inhibitions that are at least2-fold, more typically at least 10-fold, and most typically at least100-fold relative to the mammalian polymerase. The term “inhibit apolymerase” as used herein means reduction of the polymerase activity(e.g., as evidenced by formation of product on a PAGE) of at least 5%,more typically at least 35%, and most typically of at least 90% ascompared to the polymerase activity under identical conditions but inthe absence of the inhibiting compound.

[0092] Consequently, a particularly preferred use of contemplatedcompounds includes use of such compounds in the treatment of viraldiseases, and particularly HCV. Therefore, especially preferred aspectsof contemplated compounds include methods and compositions for thetreatment of hepatitis C in humans or other host animals in which aneffective amount of the contemplated compounds, pharmaceuticallyacceptable salts or prodrugs thereof are administered, optionally in apharmaceutically acceptable carrier. Where such compounds are prodrugs,it should be recognized that the prodrugs either possess antiviral(e.g., anti-HCV) activity, or are metabolized to a compound thatexhibits such activity.

[0093] In an especially preferred use, the inventors contemplate apharmaceutical composition that includes a nucleotide analog having astructure according to Formula 1 or Formula 2, wherein the nucleotideanalog is present in the composition effective to inhibit a viralpolymerase of an HCV virus or to act as a substrate for the viralpolymerase of the HCV virus

[0094] wherein Z¹ and Z² are independently H, alkyl, halogen, OR⁵, SR⁵,NR⁵R⁶, CO-alkyl, CO-aryl, or CO-alkoxyalkyl, and wherein W is H, OR⁵,SR⁵, NR⁵R⁶, NH(NR⁵R⁶), N(alkyl)(NR⁵R⁶CN, C(O)NR⁵R⁶, C(NH)NR⁵R⁶, orhalogen; wherein V is hydrogen, halogen, OR⁵, SR⁵, NR⁵R⁶, NH(NR⁵R⁶),N(alkyl)(NR⁵R⁶), CN, C(O)NR⁵R⁶, or C(NH)NR⁵R⁶, and wherein Y is H,alkyl, halogen, OR⁵, SR⁵, NR⁵R⁶, CO-alkyl, CO-aryl, or CO-alkoxyalkyl; Xis a covalent bond between the C4′-atom of the sugar and the carbon atomin the phosphonate group, O, CH₂, CHR⁵, CHHalogen, or C(Halogen)₂; D isCH₂, CHHalogen, or C(Halogen)₂; R¹ and R² are independently H,phosphate, or a group that is preferentially removed in a hepatocyte toyield a corresponding OH group; R₁′, R₂′, R₃′, and R₄′ are independentlyH or alkyl; R³ and R⁴ are H, or where at least one of R₁′, R₂′, R₃′, andR₄′ is alkyl, R³ and R⁴ are independently H, phosphate, acyl, alkyl, ora group that is preferentially removed in the hepatocyte to acorresponding C2′—OH group or C3′—OH group; R⁵ and R⁶ are independentlyH, alkyl, or acyl.

[0095] Where compounds have a structure according to Formula 1, it isparticularly preferred that X is a covalent bond between the C4′-atom ofthe sugar and the carbon atom in the phosphonate group, O, or CH₂, andwherein at least one of R₁′, R₂′, R₃′, and R₄′ is CH₃ (most preferablyR₂′ is CH₃). Alternatively, or additionally Z₁ and Z₂ is H, and whereinW is NR⁵R⁶. On the other hand, where compounds have a structureaccording to Formula 2, it is particularly preferred that X is acovalent bond between the C4′-atom of the sugar and the carbon atom inthe phosphonate group, O, or CH₂, and wherein at least one of R₁′, R₂′,R₃′, and R₄′ is CH₃ (most preferably R₂′ is CH₃). Alternatively, oradditionally Y is H or CH₃, and wherein V is OH, or NR⁵R⁶.

[0096] Contemplated compounds can be administered as any salt orprodrug, that upon administration to the recipient is capable ofproviding directly or indirectly the parent compound, or that exhibitsactivity itself. Non-limiting examples are the pharmaceuticallyacceptable salts (alternatively referred to as “physiologicallyacceptable salts”), and a compound that has been alkylated or acylatedat the 5′-position or on the purine or pyrimidine base (a type of“pharmaceutically acceptable prodrug”). Further, the modifications canaffect the biological activity of the compound, in some cases increasingthe activity over the parent compound. This can easily be assessed bypreparing the salt or prodrug and testing its antiviral activityaccording to the methods described herein or other methods known tothose skilled in the art.

[0097] Where contemplated nucleosides are administered in apharmacological composition, it is contemplated that suitable compoundscan be formulated in a mixture with a pharmaceutically acceptablecarrier. For example, contemplated compounds can be administered orallyas pharmacologically acceptable salts, or intravenously in aphysiological saline solution (e.g., buffered to a pH of about 7.2 to7.5). Conventional buffers such as phosphates, bicarbonates or citratescan be used for this purpose. Of course, one of ordinary skill in theart may modify the formulations within the teachings of thespecification to provide numerous formulations for a particular route ofadministration. In particular, contemplated nucleosides may be modifiedto render them more soluble in water or other vehicle, which forexample, may be easily accomplished by minor modifications (saltformulation, esterification, etc.) that are well within the ordinaryskill in the art. It is also well within the ordinary skill of the artto modify the route of administration and dosage regimen of a particularcompound in order to manage the pharmacokinetics of the presentcompounds for maximum beneficial effect in a patient. Thus, and amongvarious other administrations, it is preferred that contemplatedpharmaceutical compositions will be administered to a viral polymerasewhich is disposed in a cell infected with HCV (most typically, the cellis disposed in a patient infected with HCV).

[0098] In yet another especially preferred aspect, the active compoundcan be administered in combination or alternation with another anti-HCVagent, which may be a nucleoside analog (e.g., ribavirin, Viramidine)and/or an interferon (e.g., pegylated IFN-alpha or IFN-gamma). Incombination therapy, an effective dosage of two or more agents, areadministered together, whereas during alternation therapy an effectivedosage of each agent is administered serially. The dosages will dependon absorption, inactivation, and excretion rates of the drugs as well asother factors known to those skilled in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens and schedules should be adjusted overtime according to the individual need and the professional judgement ofthe person administering or supervising the administration of thecompositions.

[0099] Consequently, methods of treatment of a viral or bacterialinfection will include one step in which it is ascertained that thebacterium or virus includes a polymerase that incorporates compoundsaccording to the inventive subject matter into a nascent polynucleotide.In a further step, contemplated compounds are administered to a mammalinfected with the virus or bacterium in a dosage effective to inhibitthe polymerase. Alternatively, contemplated compounds may beadministered to a polymerase in vitro or in vivo at a concentrationeffective to inhibit the polymerase or at a concentration effectivce toact as a substrate for the polymerase.

[0100] In still further contemplated aspects, nucleosides can bescreened for their ability to inhibit HCV polymerase activity in vitroaccording to screening methods set forth more particularly herein. Onecan readily determine the spectrum of activity by evaluating thecompound in the assays described herein or with another confirmatoryassay.

[0101] In one especially preferred aspect, the efficacy of the anti-HCVcompound is measured according to the concentration of compoundnecessary to reduce the virus replication in vivo, by 50% (i.e. thecompound's EC₅₀) according to methods set forth more particularlyherein. For example, the efficacy of an anti-HCV compound may bemeasured according to the concentration of compound necessary to reducethe virus replication in vivo, by 50% (i.e. the compound's EC₅₀)according to the method set forth more particularly herein. Inparticularly preferred aspects, the compound exhibits an EC₅₀ of lessthan 100, 50, 25, 15, 10, 5 or 1 micromolar.

[0102] Alternatively, nucleosides can be screened for their ability toinhibit HCV polymerase activity in vitro according to the screeningmethods set forth more particularly herein. One can readily determinethe spectrum of activity by evaluating the compound in the assaysdescribed herein or with another confirmatory assay.

Synthesis of Contemplated Compounds

[0103] It should generally be recognized that nucleoside phosphonatesaccording to the inventive subject matter may be synthesized usingnumerous protocols known to those skilled in the art. However, in apreferred procedure, synthesis of the nucleoside phosphonates may beachieved by condensing the phosphonate group to the modified nucleoside.

[0104] General Synthesis of 5′-Deoxy-5′-phosphonate Nucleosides

[0105] Synthesis can be achieved by the modification of a preformednucleoside as described in the general exemplary scheme below, and itshould be specially pointed out that various sugar modifications may beintroduced in the compounds according to the inventive subject matterusing modified sugars before coupling of the sugar with the base, or bymodifying the sugar portion in the nucleoside. Similarly, theheterocyclic base may be modified at any appropriate step in thesynthesis of contemplated compounds.

[0106] In a particularly preferred aspect, the synthesis of a5′-deoxy-5′-phosphonate nucleoside is achieved by the derivatization ofthe 5′-position of the preformed nucleoside as depicted in FIG. 1. Atypical synthesis may be carried out by protecting 2′- and 3′-alcohols,and then converting the 5′-alcohol of the nucleoside into a 5′-bromoderivative by the reaction with carbon tetrabromide andtriphenylphosphine. The Michaelis-Arbuzov reaction of the protected5′-bromo nucleoside with triethyl phosphite yields the corresponding5′-phosphonate nucleoside. Removal of the ethyl group by treatment withbromotrimethylsilane, followed by the deprotection of the benzoyl groupprovides the target compound. Alternatively, synthesis may also beachieved by modification of a sugar and condensation of the sugarphosphonate with the desired base as schown below in FIG. 1A.

[0107] In a particular preferred aspect, synthesis of the5′-deoxy-5′-phosphonate nucleoside is accomplished by the derivatizationof the 5-position of sugar to make 5-phosphonate sugar and thesubsequent glycosylation of the sugar phosphonate with variousheterocyclic bases as depicted in FIG. 2. Here,methyl-2,3-O-isopropylidene-β-D-ribofuranose is readily converted to its5-bromo derivative in high yield. After the activation of an anomericposition, the Michaelis-Arbuzov reaction is performed to yield the5-phosphonate sugar derivative. 6-Chloropurine is silylated and thencondensed with the 5-phosphonate sugar in the presence of trimethylsilyltriflate to afford the protected 5′-deoxy-5′-phosphonate nucleoside.Deprotection with bromotrimethylsilane provides the target compound.

[0108] General Synthesis of 5′-Deoxy-5′-methylphosphonate Nucleosides

[0109] The synthesis of 5′-deoxy-5′-methylphosphonate nucleosides may beachieved by modification of a preformed nucleoside as described inexemplary FIG. 3. In a particularly contemplated embodiment, thesynthesis of the 5′-deoxy-5′-methyl phosphonate nucleoside isaccomplished by the reaction of the preformed nucleoside 5′-aldehydewith a stabilized Wittig reagent as depicted in FIG. 3A below. To modifythe 5′-position of a nucleoside it is necessary to protect any reactivegroups other than 5′-alcohol. Treatment of the protected nucleoside withdicyclohexylcarbodiimide and dimethyl sulfoxide in the presence ofpyridium trifluoroacetate produces a 5′-aldehyde, which is treated withWittig reagent, diphenyl triphenylphosphoranylidenemethylphosphonate, tomake a vinyl phosphonate. Catalytic hydrogenation of the vinylphosphonate with Pd—C gives a saturated phosphonate. Transesterificationwith sodium benzoxide, deprotection and palladium-catalyzedhydrogenolysis of benzyl ester provides the desired 5′-deoxy-5′-methylphosphonate nucleoside.

[0110] Alternatively, the synthesis may also be achieved by modificationof a sugar and condensation of the sugar phosphonate with the desiredbase as shown on the general example in FIG. 3B.

[0111] In a particularly preferred aspect, the 5′-deoxy-5′-methylphosphonate nucleoside can be prepared starting from ribo-hexofuranoseas shown in FIG. 4. Application of the similar strategy described inFIG. 2, i.e. bromination-Arbuzov reaction-glycosylation-deprotectionsequence, affords the target nucleoside phosphonate. Alternatively, thesugar phosphonate can be prepared in a slightly different manner, asdescribed in FIG. 4A.

[0112] In yet another particularly preferred aspect, synthesis of aphosphonate isostere of a nucleoside monophosphate is achieved by using2′-deoxyadenosine as a starting material as depicted in FIG. 5. Atypical synthesis is carried out by oxidizing the 5′-hydroxyl group of2′-deoxyadenosine with potassium permanganate to give the corresponding5′-carboxylic acid. Decarboxylative elimination of the carboxylic acidprovides furanoid glycal. Regio- and stereo-selective addition ofdimethyl (hydoxymethyl)phosphonate to a double bond of the furanoidglycal is mediated by iodine monobromide.1,8-diazabicyclo[5,5,0]undec-7-ene-catalyzed elimination of theresulting iodide intermediate provides 2,5-dihydrofuran derivative.Bis-hydroxylation on the double bond of the dihydrofuran with osmiumtetraoxide and 4-methyl-morpholine-N-oxide, followed by removal ofprotecting groups provides the target compound.

[0113] Alternatively, a similar synthetic strategy can be applied towardthe synthesis of the phosphonate analogue containing methyl group at the2′ position of the sugar as shown in FIG. 6 below. Protection of 3′- and5′-hydroxyl groups of adenosine by silylation, followed by oxidation ofthe 2′-hydroxyl with dicyclohexylcarbodiimide and dimethyl sulfoxideaffords the ketone derivative. Wittig reaction of the ketone derivativewith methyltriphenylphosphonium bromide provides the corresponding2′-methylideneadenosine. Hydrogenation in the presence of Pd—C, followedby desilylation affords 2′-deoxy-2′-methyladenosine, can be convertedinto the desired phosphonate isostere by repeating the same sequencedescribed in FIG. 5.

[0114] In a still further preferred aspect of the inventive subjectmatter, the synthesis of a carboxylic phosphonate is described asindicated in FIG. 7 below. Here, the carbocyclic nucleoside can beformed directly by palladium-catalyzed reaction of a sodium salt of6-chloropurine with the enantiomerically pure allylic acetate.Subsequent amination of the 6-chloro nucleoside gives an adeninederivative. It is then treated withp-tolylsulfonyloxy-methanephosphonate in the presence of a base toprovide the corresponding nucleoside phosphonate. Removal of the ethylgroup by treatment of bromotrimethylsilane, followed bybis-hydroxylation with osmium tetraoxide affords the target compound.

EXAMPLES

[0115] 9-[5′-Deoxy-5′-(dihyoxyphosphinyl)-β-D-ribo-furanosyl]adenine(FIG. 2)

[0116] Methyl 2,3-di-O-benzoyl-5-bromo-5-deoxy-d-ribofuranoside: Asolution of methyl(5-deoxy-5-bromo-2,3-O-isopropylidene-)-β-D-ribofuranose (100 g, 0.4mol) in MeOH (600 mL) was treated with 0.05 N aqueous sulfuric acid (200mL) and refluxed for 10 h. The solution was neutralized with saturatedNaHCO₃ and the volatiles were removed under reduced pressure. Thesolution was extracted with EtOAc (10×100 mL) and the combined organicsolution was washed with brine (500 mL), dried with Na₂SO₄, andconcentrated to dryness. The resulting yellow oil was dissolved inpyridine (500 mL) and treated with benzoyl chloride (118 g, 0.84 mol)and the reaction was stirred at room temperature for 20 h. The mixturewas diluted with slow addition of H₂O (500 mL) and extracted with ether(5×200 mL). The combined organic solution was washed with 3N ice-coldsulfuric acid (5×200 mL) and brine (2×200 mL), dried with Na₂SO₄, andconcentrated to dryness. Silica gel chromatography (EtOAc:Hexanes=40:60)yielded 127 g of the target compound (75%).

[0117] 2,3-Di-O-benzoyl-5-bromo-5-deoxyribofuranosyl acetate: Thecompound obtained in step a.) (127 g, 0.29 mol) in dioxane (1300 mL) wasrefluxed with 1 N HCl for 22 h. The solution was neutralized withsaturated NaHCO₃ and the volatiles were removed under reduced pressure.The mixture was extracted with EtOAc (10×100 mL) and the combinedorganic solution was washed with brine (500 mL), dried with Na₂SO₄, andconcentrated to dryness. The red syrup was dissolved in pyridine (200mL) and treated with acetyl chloride (50 mL) and DMAP (1 g) and thereaction was stirred at room temperature for 20 h. The mixture wasdiluted with slow addition of H₂O (200 mL) and extracted with CH₂Cl₂(5×200 mL). The combined organic solution was washed with 3N ice-coldsulfuric acid (5×100 mL) and brine (2×100 mL), dried with Na₂SO₄, andconcentrated to dryness. Silica gel chromatography (EtOAc:Hexanes=40:60)yielded 68 g of the target compound (50%).

[0118]1-O-Acetyl-2,3-di-O-benzoyl-5-deoxy-5-(diethoxyphosphinyl)-D-ribofuranose:2,3-Di-O-benzoyl-5-bromo-5-deoxyribofuranosyl acetate (68.0 g, 0.143mol) was dissolved in triethyl phosphite (205 mL) and the solution washeated under reflux for 30 h. The volatiles were removed under reducedpressure and the resulting oil was dissolved in ether (780 mL). Theorganic solution was washed with brine (2×100 mL), dried with Na₂SO₄,and concentrated to dryness. Silica gel chromatography(EtOAc:Hexanes=80:20) yielded 59.4 g of the target compound (78%).

[0119]9-[5′-Deoxy-2′,3′-O-benzoyl-5′-(diethoxyphosphinyl)-β-D-ribofuranosyl]-6-chloropurine:6-Chloropurine (1.75 g, 10 mmol) was mixed with HMDS (50 mL) andrefluxed with ammonium sulfate (0.1 g) for 4 h. The mixture wasevaporated and dried in vacuo for 2 h. The solid was dissolved in CH₃CN(10 mL) and treated with the compound obtained in step c.) in CH₃CN (10mL) followed by the additon of TMSOTf (6.67 g, 30 mmol). After 24 h atroom temperature, the mixture was poured into saturated NaHCO₃. Themixture was extracted with EtOAc (10×25 mL) and the combined organicsolution was washed with brine (100 mL), dried with Na₂SO₄, andconcentrated to dryness. Silica gel chromatography(CH2Cl2:Acetone=80:20) yielded 3.69 g of the target compound (60%).

[0120] 9-[5′-deoxy-5′-(dihyoxyphosphinyl)-β-D-ribo-furanosyl]adenine:The compound obtained in step d.) (3.07 g, 5 mmol) in CH₃CN (10 mL) wastreated with bromotrimethylsilane (2.3 g, 15 mmol) and stirred for 6 h.The volatiles were removed under reduced pressure, and the crude wasdissolved in methanolic ammonia (50 mL) and heated in a bomb at 80° C.for 16 h. The solution was concentrated to 10 mL and 10 mL of H₂O wasadded and extracted with CH₂Cl₂ (3×20 mL). The organic layer was washedwith H₂O and the combined H₂O solution was chroinatographed on theDEAE-cellulose column with ammonium bicarbonate. Concentration yielded0.93 g of the target compound (51%).

[0121]9-[5′,6′-Dideoxy-6′-(hydroxyphosphinyl)-β-D-ribo-hexofuranosyl)adenine(FIG. 4)

[0122] 9-(5′-O-(4,4′-dimethoxytrityl-β-D-ribofuranosyl)adenine: To asuspension of adenosine (1) (29.0 g, 113 mmol), which was dried in vacuofor 8 h at 80° C. with P₂O₅, in a mixture of DMF (900 mL) and pyridine(100 mL) was added 4,4′-dimethoxytrityl chloride (41.9 g, 121 mmol). Thereaction mixture was stirred at room temperature for 2 days and quenchedby the addition of H₂O (50 mL). After stirring for 1 h, the mixture wasevaporated and the resulting slurry was poured into saturated NaHCO₃solution (1.5 L). The white precipitate was collected by vacuumfiltration and washed with cold H₂O (200 mL). The resulting solid wasdried and recrystallized in ethyl acetate-benzene mixture to yield 27.3g (44%) of 9-(5′-O-(4,4′-dimethoxytrityl-β-D-ribofuranosyl)adenine as awhite solid.

[0123] N⁶-Benzoyl-9-(2′,3′-di-O-benzoyl-β-D-ribofuranosyl)adenine: To asolution of 9-(5′-O-(4,4′-dimethoxytrityl-β-D-ribofuranosyl)adenine(17.1 g, 30.2 mmol) in anhydrous pyridine (250 mL) was added benzoicanhydride (27.3 g, 120.8 mmol). The reaction mixture was heated underreflux for 3 h. The excess benzoic anhydride was quenched by the additonof MeOH (20 mL), followed by a 15 min reflux period. The mixture wasconcentrated to dryness and dissolved in CHCl₃ (500 mL). The organicsolution was washed with saturated NaHCO₃ (2×300 mL) and brine (1×300mL), dried with Na₂SO₄, and concentrated to dryness. The crudebenzoylated product was treated with a 2;2; 1 mixture of H₂O-formicacid-THF (500 mL). The solution was stirred at room temperature for 1 hand neutralized with the addition of 50% aqueous NaOH solution. Thesolution was extracted with CHCl₃ (3×200 mL) and the combined organicsolution was washed with brine (1×100 mL), dried with Na₂SO₄, andconcentrated to dryness. Recrystallization in ethanol yielded 8.2 g(82%) of the target compound as a white solid.

[0124]N⁶-Benzoyl-9-[2′,3′-di-O-benzoyl-5′,6′-dideoxy-6′-(diphenoxyphosphinyl)-β-D-ribo-hex-5-enofuranosyl]adenine:To a solution of the compound obtained in step b.) (1.65 g, 2.84 mmol),1,3-dicyclohexylcarbodiimide (2.34 g, 11.4 mmol) and pyridine (0.32 mL)in anhydrous DMSO (15 mL) was added trifluoroacetic acid (0.15 mL) andthe mixture was stirred at room temperature for 22 h. To the mixture wasadded diphenyl (triphosphoranylidene)methylphosphonate (2.89 g, 5.68mmol) and stirring was continued for 24 h. Excess carbodiimide washydrolyzed by a careful addition of a solution of oxalic acid dihydrate(771 mg, 8.56 mmol) in MeOH (3 mL). After stirring for 10 min, thevolatiles were removed under reduced pressure and the urea formed wasremoved by vacuum filtration. The filtrate was extracted with EtOAc (300mL) and the organic solution was washed with brine (2×100 mL), driedwith Na₂SO₄, and concentrated to dryness. Silica gel chromatography(EtOAc) yielded 1.43 g of the target compound (62%).

[0125]N⁶-Benzoyl-9-[2′,3′-di-O-benzoyl-5′,6′-dideoxy-6′-(diphenoxyphosphinyl)-β-D-ribo-hexofuranosyl]adenine:To a solution of the vinylphosphonate (800 mg, 0.99 mmol) in MeOH (30mL) was added Pd—C (10%, 300 mg) and the reaction mixture was stirredunder 30 psi of hydrogen. After 20 h, the mixture was filtered throughCelite to remove the catalyst and the filtrate was evaporated todryness. Silica gel chromatography (EtOAc:Hexanes=75:25) yielded 410 mgof the target compound (51%).

[0126]N⁶-benzoyl-9-[5′,6′-dideoxy-6′-(dibenzyloxyphosphinyl)-β-D-ribo-hexofuranosyl]adenine:Sodium hydride (100 mg, 2.5 mmol) was added to 5 mL of benzyl alcoholand the resulting suspension was stirred for 30 min. To the mixture wasadded a solution of the compound obtained in step d.) (360 mg, 0.44mmol) in anhydrous DMSO (5 mL). The reaction was stirred at roomtemperture for 4 h and quenched with saturated NH₄Cl solution (20 mL).The mixture was extracted with EtOAc (2×30 mL) and the combined organicsolution was washed with brine (30 mL), dried with Na₂SO₄, andconcentrated to dryness. Silica gel chromatography (EtOAc:MeOH=90:10)yielded 190 mg ofN⁶-benzoyl-9-[5′,6′-dideoxy-6′-(dibenzyloxyphosphinyl)-β-D-ribo-hexofuranosyl]adenine(69%).

[0127]9-[5′,6′-dideoxy-6′-(dibenzyloxyphosphinyl)-β-D-ribo-hexofuranosyl]adenine:A solution of the compound obtained in step e.) (190 mg, 0.30 mmol) inmethanolic ammonia (40 mL, saturated at 0° C.) was stirred at roomtemperature in a sealed bomb for 24 h. The bomb was cooled to 0° C.before opening. The reaction mixture was stirred at RT for 1 h and thenconcentrated to dryness. Silica gel chromatography (CH₂Cl₂:MeOH=90:10)yielded 146 mg of the target compound (92%) as a pale foam.

[0128]9-[5′,6′-dideoxy-6′-(hydroxyphosphinyl)-β-D-ribo-hexofuranosyl]adenine:To a solution of the compound obtained in step d.) (146 mg, 0.28 mmol)in MeOH (20 mL) was added Pd—C (10%, 150 mg) and the reaction mixturewas stirred under hydrogen at atmospheric pressure. After 16 h, themixture was filtered through Celite to remove the catalyst and thefiltrate was evaporated to dryness. The resulting solid obtained waswashed with CH₂Cl₂ (1 mL) to yield 53 mg of the target compound (55%) asa white solid.

[0129] (2R, 3R, 4S,5R)-9-[Tetrahydro-3,4-dihydroxy-5-(phosphonomethoxy)-2-furanyl]adenine(FIG. 5)

[0130] 2′-Deoxyadenosine-5′-carboxylic acid: To a solution of2′-deoxyadenosine (8.60 g, 32 mol) in H₂O (1 L) was added dropwisesolutions of KMnO₄ (15.0 g, 95 mmol) ) in H₂O (1 L) and KOH (4.0 g, 71mmol) ) in H₂O (500 mL). The reaction mixture was stirred at roomtemperature for 3 days and an excess KMnO₄ was quenched with a slowaddition of 30% H₂O₂. The mixture was filtered under reduced pressureand the filtrate was concentrated to 200 mL and the resulting solutionwas acidified to pH 4.5 with 3N HCl. The precipitate was filtered anddried in vacuo at 40° C. for 8 h to yield 2.53 g of the target compound(30%) as a white solid.

[0131] 9-[2,3-Dihydro-2(R)-furanyl-N⁶-pivaloyladenine: To a suspensionof the compound obtained in step a.) (2.53 g, 8.9 mol) in DMF (100 mL)was added N,N-dimethylformamide dineopentyl acetal (7.6 mL, 29 mmol).The reaction mixture was stirred at 95° C. for 16 h and concentrated todryness. The residue was dissolved in a mixture of MeOH (65 mL) andconcentrated NH₄OH (65 mL) and stirred at room temperature for 24 h. Thevolatiles were removed under reduced pressure. Silica gel chromatography(CH₂Cl₂:MeOH=90:10) yielded 1.31 g of 9-[2,3-dihydro-2(R)-furanyladenine(61%) as a white solid. To a solution of the compound obtained above(1.70 g, 8.4 mol), pyridine (0.82 mL, 10.2 mmol) and4-dimethylaminopyridine (145 mg, 1.2 mmol) in 1,2-dichloroethane (60 mL)was added pivaloyl chloride (1.22 mL, 9.9 mmol). The reaction mixturewas stirred at 65° C. for 16 h and diluted with CH₂Cl₂ (300 mL). Theorganic solution was washed with brine (500 mL), dried with Na₂SO₄, andconcentrated to dryness. Silica gel chromatography (CH₂Cl₂:MeOH=97:3)yielded 1.30 g of the target compound (51%).

[0132] (2R,5R)-N⁶-Pivaloyl-9-[2,5-Dihydro-5-[(dimethoxyphosphinyl)methoxy]-2-furanyl]adenine:To a solution of the compound obtained in the previous step (980 mg, 3.4mol) and dimethyl(hydroxymethyl)phosphonate (1.71 g, 13.6 mmol) inCH₂Cl₂ (8 mL) at −25° C. was added dropwise a solution of iodinemonobromide (1.41 g, 6.8 mmol) ) in CH₂Cl₂ (8 mL). The reaction mixturewas stirred at −25° C. for 8 h and diluted with CH₂Cl₂ (100 mL). Theorganic solution was washed with aqueous NaHCO₃ (50 mL), aqueous sodiumbisulfite (2×50 mL) and brine (500 mL), dried with Na₂SO₄, andconcentrated to dryness. Silica gel chromatography (CH₂Cl₂:MeOH=95:5)yielded 1.70 g of (2R, 4S,5R)-N⁶-pivaloyl-9-[tetrahydro-4-iodo-5-[(dimethoxyphosphinyl)methoxy]-2-furanyl]adenine(90%). To a solution of the compound obtained above (811 mg, 1.5 mmol)THF (10 mL) was added 1,8-diazabicyclo[5,5,0]undec-7-ene (0.44 mL, 2.9mmol). The reaction mixture was stirred at 60° C. for 5 h and thevolatiles were removed under reduced pressure. The residue was dissolvedin CH₂Cl₂ (300 mL) and the organic solution was washed with brine (200mL), dried with Na₂SO₄, and concentrated to dryness. Silica gelchromatography (CH₂Cl₂:MeOH=95:5) yielded 381 mg of the target compound(61%).

[0133] (2R, 3R, 4S,5R)-N⁶-Pivaloyl-9-[tetrahydro-3,4-dihydroxy-5-[(dimethoxyphosphinyl)methoxy]-2-furanyl]adenine:To a solution of the compound obtained in the previous step (178 mg,0.42 mol) and 4-methylmorpholine N-oxide (0.11 g, 0.63 mmol) in acetone(6 mL) and t-BuOH (1 mL) OsO₄ (0.26 ml, 0.020 mmol) was added. Thereaction mixture was stirred at room temperature for 16 h and thevolatiles were removed under reduced pressure. The residue was dissolvedin EtOAc (100 mL) and the organic solution was washed with aqueoussodium thiosulfate (2×50 mL) and brine (50 mL), dried with Na₂SO₄, andconcentrated to dryness. Silica gel chromatography (CH₂Cl₂:MeOH=90:10)yielded 145 mg of the target compound (75%).

[0134] (2R, 3R, 4S,5R)-9-[Tetrahydro-3,4-dihydroxy-5-(phosphonomethoxy)-2-furanyl]adenine:To the compound obtained in the previous step (1.10 g, 2.8 mmol) in MeOH(15 mL) at 0° C. was added 25% sodium methoxide in MeOH (2.5 mL). Thereaction mixture was stirred at room temperature for 7 h and neutralizedto pH 8.0 by addition of 2 N HCl. The volatiles were removed underreduced pressure. The crude mixture was dissolved in DMF (15 mL) andtreated with bromotrimethylsilane (3.5 mL) at 0° C. After stirring atroom temperature for 6 h, the volatiles were removed in vacuo. The oilwas diluted with concentrated NH₄OH (1.5 mL) and reevaporated. C₁₈reverse-phase chromatography with water yielded 0.55 g of the targetcompound (65%).

[0135] (2R, 3R, 4S,5R)-9-[Tetrahydro-3,4-dihydroxy-3-methyl-5-(phosphonomethoxy)-2-furanyl]adenine(FIG. 6)

[0136]3′,5′-O-(Tetraisopropyldisilyloxane-1,3-diyl)-2′-deoxy-2′-methylideneadenosine:To a suspension of adenosine (10.8 g, 40 mmol) in pyridine (1000 mL) at0° C. was added 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (15.5 mL,48 mmol). The reaction mixture was stirred at room temperature for 2days and quenched by an addition of ethanol (5 mL). The mixture wasevaporated and the resulting oil was diluted with EtOAc (1000 mL). Theorganic solution was washed with aqueous NaHCO₃ (2×500 mL) and brine(500 mL), dried with Na₂SO₄, and concentrated to dryness. Silica gelchromatography (CH₂Cl₂:MeOH=90:10) yielded 18 g of3′,5′-O-(1,1,3,3,-tetraisopropyl-disiloxane-1,3-diyl)-adenosine (88%).

[0137] To a solution of the compound obtained above (2.5 g, 5.0 mmol),1,3-dicyclohexylcarbodiimide (1.81 g, 8.8 mmol) and pyridine (0.48 mL)in anhydrous DMSO (15 mL) was added trifluoroacetic acid (0.23 mL). Themixture was stirred at room temperature for 22 h and excess carbodiimidewas hydrolyzed by a careful addition of a solution of oxalic aciddihydrate in MeOH. After stirring for 10 min, the volatiles were removedunder reduced pressure and the urea formed was removed by vacuumfiltration. The filtrate was extracted with EtOAc (300 mL) and theorganic solution was washed with brine (2×100 mL), dried with Na₂SO₄,and concentrated to dryness. The resulting crude was dissolved inbenzene (15 mL) and the solution was cooled down to −78° C. The mixturewas added to a solution of PPh₃MeBr (3.57 g, 10 mmol) and sodiumpentoxide (0.99 g, 9 mmol) in ether (125 mL), which was pre-stirred atroom temperature for 2 h. After the addition, the reaction mixture wasslowly warmed to 4° C. at which temperature the mixture was kept for 48h. The reaction mixture was washed with brine (2×100 mL), dried withNa₂SO₄, and concentrated to dryness. Silica gel chromatography (CH₂Cl₂)yielded 1.52 g of the target compound (60%).

[0138] 2′-Deoxy-2′-methyladenosine: To a solution of the compoundobtained in the previous step (320 mg, 1.1 mmol) in MeOH (44 mL) wasadded Pd—C (10%, 200 mg) and the reaction mixture was stirred under 5psi of hydrogen. After 4 h, the mixture was filtered through Celite toremove the catalyst and the filtrate was evaporated to dryness. Silicagel chromatography (CH₂Cl₂:MeOH=90:10) yielded 240 mg of the reducedcompound (75%). To a solution of the reduced compound (240 mg, 0.82mmol) in THF (20 mL) was added 1 M solution of TBAF (2.5 mL, 2.5 mmol)in THF. The reaction mixture was stirred at room temperature for 1 h andthe volatiles were removed under reduced pressure. Silica gelchromatography (CH₂Cl₂:MeOH=80:20) yielded 196 mg of the target compound(90%).

[0139] 2′-Deoxy-2′-methyladenosine-5′-carboxylic acid: To a solution ofthe compound obtained in the previous step (8.60 g, 32 mol) in H₂O (1 L)was added dropwise solutions of KMnO₄ (15.0 g, 95 mmol) ) in H₂O (1 L)and KOH (4.0 g, 71 mmol) ) in H₂O (500 mL). The reaction mixture wasstirred at room temperature for 3 days and excess KMnO₄ was quenchedwith a slow addition of 30% H₂O₂. The mixture was filtered under reducedpressure and the filtrate was concentrated to 200 mL and the resultingsolution was acidified to pH 4.5 with 3N HCl. The precipitate wasfiltered and dried in vacuo at 40° C. for 8 h to yield.2.53 g of thetarget compound (30%) as a white solid.

[0140] 9-[2,3-Dihydro-3-methyl-2(R)-furanyl-N⁶-pivaloyladenine: To asuspension of the compound obtained in the previous step (2.53 g, 8.9mol) in DMF (100 mL) was added N,N-dimethylformamide dineopentyl acetal(7.6 mL, 29 mmol). The reaction mixture was stirred at 95° C. for 16 hand concentrated to dryness. The residue was dissolved in a mixture ofMeOH (65 mL) and concentrated NH₄OH (65 mL) and stirred at roomtemperature for 24 h. The volatiles were removed under reduced pressure.Silica gel chromatography (CH₂Cl₂:MeOH=90:10) yielded 1.31 g of9-[2,3-dihydro-3-methyl-2(R)-furanyladenine (61%) as a white solid. To asolution of the compound obtained above (1.70 g, 8.4 mol), pyridine(0.82 mL, 10.2 mmol) and 4-dimethylaminopyridine (145 mg, 1.2 mmol) in1,2-dichloroethane (60 mL) was added pivaloyl chloride (1.22 mL, 9.9mmol). The reaction mixture was stirred at 65° C. for 16 h and dilutedwith CH₂Cl₂ (300 mL). The organic solution was washed with brine (500mL), dried with Na₂SO₄, and concentrated to dryness. Silica gelchromatography (CH₂Cl₂:MeOH=97:3) yielded 1.30 g of the target compound(51%).

[0141] (2R,5R)-N⁶-Pivaloyl-9-[2,5-Dihydro-5-[(dimethoxyphosphinyl)methoxy]-3-methyl-2-furanyl]adenine:To a solution of the compound obtained in the previous step (980 mg, 3.4mol) and dimethyl(hydroxymethyl)phosphonate (1.71 g, 13.6 mmol) inCH₂Cl₂ (8 mL) at −25° C. was added dropwise a solution of IBr (1.41 g,6.8 mmol) ) in CH₂Cl₂ (8 mL). The reaction mixture was stirred at −25°C. for 8 h and diluted with CH₂Cl₂ (100 mL). The organic solution waswashed with, aqueous NaHCO₃ (50 mL), aqueous sodium bisulfite (2×50 mL)and brine (500 mL), dried with Na₂SO₄, and concentrated to dryness.Silica gel chromatography (CH₂Cl₂:MeOH=95:5) yielded 1.70 g of (2R, 4S,5R)-N⁶-pivaloyl-9-[tetrahydro-4-iodo-5-[(dimethoxyphosphinyl)methoxy]-3-methyl-2-furanyl]adenine(90%).

[0142] To a solution of the compound obtained above (811 mg, 1.5 mmol)in THF (10 mL) was added 1,8-diazabicyclo[5,5,0]undec-7-ene (0.44 mL,2.9 mmol). The reaction mixture was stirred at 60° C. for 5 h and thevolatiles were removed under reduced pressure. The residue was dissolvedin CH₂Cl₂ (300 mL) and the organic solution was washed with brine (200mL), dried with Na₂SO₄, and concentrated to dryness. Silica gelchromatography (CH₂Cl₂:MeOH=95:5) yielded 381 mg of the target compound(61%).

[0143] (2R, 3R, 4S,5R)-N⁶-Pivaloyl-9-[tetrahydro-3,4-dihydroxy-5-[(dimethoxyphosphinyl)methoxy]-3-methyl-2-furanyl]adenine:To a solution of the compound obtained in the previous step (178 mg,0.42 mol) and 4-methylmorpholine N-oxide (0.11 g, 0.63 mmol) in acetone(6 mL) and t-BuOH (1 mL) was added OsO₄ (0.26 ml, 0.020 mmol). Thereaction mixture was stirred at room temperature for 16 h and thevolatiles were removed under reduced pressure. The residue was dissolvedin EtOAc (100 mL) and the organic solution was washed with aqueoussodium thiosulfate (2×50 mL) and brine (50 mL), dried with Na₂SO₄, andconcentrated to dryness. Silica gel chromatography (CH₂Cl₂:MeOH=90:10)yielded 145 mg of the target compound (75%).

[0144] (2R, 3R, 4S,5R)-9-[Tetrahydro-3,4-dihydroxy-3-methyl-5-(phosphonomethoxy)-2-furanyl]adenine:To the compound obtained in the previous step (1.10 g, 2.8 mmol) in MeOH(15 mL) at 0° C. was added 25% sodium methoxide in MeOH (2.5 mL). Thereaction mixture was stirred at room temperature for 7 h and neutralizedto pH 8.0 by an addition of 2 N HCl. The volatiles were removed underreduced pressure. The crude was dissolved in DMF (15 mL) and was treatedwith bromotrimethylsilane (3.5 mL) at 0° C. After stirring at roomtemperature for 6 h, the volatiles were removed in vacuo. The oil wasdiluted with concentrated NH₄OH (1.5 mL) and reevaporated. C₁₈reverse-phase chromatography with water yielded 0.55 g of the targetcompound (65%).

[0145] (1R, 2R, 3S,4S)-9-[2,3-Dihydroxy-4-(phosphonomethoxy)-cyclopent-1-yl]adenine (FIG.7)

[0146] (1R, 4S)-9-(4-Hydroxycyclopent-2-en-1-yl)-9H-adenine: Asuspension of 6-chloropurine (1.55 g, 10 mmol) and NaH (288 mg, 12 mmol)in THF (20 mL) was stirred at room temperature for 1.5 h. To the mixturewas added a solution of (1R, 3S)-(+)-cyclopentene-1,3-diol 1-acetate(1.42 g, 10 mmol), Pd (PPh₃)₄ (1.15 g, 1.0 mmol) and PPh₃ (787 mg, 3.0mmol) in THF (10 mL), and the reaction mixture was stirred at 70° C. for18 h. The mixture was filtered through Celite and the volatiles wereremoved under reduced pressure. A solution of the crude material inmethanolic ammonia (100 mL, saturated at 0° C.) was stirred at 100° C.in a sealed bomb for 3 h. The bomb was cooled to 0° C. before opening.The reaction mixture was stirred at room temperature for 1 h and thenconcentrated to dryness. Silica gel chromatography (CH₂Cl₂:MeOH=90:10)yielded 869 mg of the target compound (40%) as a pale foam.

[0147] (1R, 4S)-9-[4-(Phosphonomethoxy)-cyclopent-2-en-1-yl]adenine: Toa solution of the compound obtained in the previous step (543 mg, 2.5mmol) and NaH (150 mg, 3.8 mmol) in DMF (50 mL) was added a solution of(EtO)(HO)POCH₂OTs (735 mg, 2.5 mmol) in DMF. After 16 h, the mixture wasneutralized with AcOH and the volatiles were removed under reducedpressure. The crude mixture was dissolved in DMF (15 mL) and treatedwith bromotrimethylsilane (3.5 mL) at 0° C. The reaction mixture wasstirred at room temperature for 6 h and the volatiles were removed underreduced pressure. The oil was diluted with concentrated NH₄OH (1.5 mL)and reevaporated. C₁₈ reverse-phase chromatography with water yielded156 mg of the target compound (20%).

[0148] (1R, 2R, 3S,4S)-9-[2,3-Dihydroxy-4-(phosphonomethoxy)-cyclopent-1-yl]adenine: To asolution of the compound obtained in the previous step (70 mg, 0.23 mol)and 4-methylmorpholine N-oxide (0.11 g, 0.63 mmol) in acetone (6 mL) andt-BuOH (1 mL) was added OsO₄ (0.26 ml, 0.020 mmol). The reaction mixturewas stirred at room temperature for 16 h and the volatiles were removedunder reduced pressure. C₁₈ reverse-phase chromatography with wateryielded 32 mg of the target compound (40%).

Biological Data

[0149] Among other nucleotide analogs (data not shown), two exemplaryphosphonate analogs of ATP, 5′-deoxy-5′-phosphonate (5′d-ATP) and5′-deoxy-5′-methylene phosphonate (5′d-CH₂-ATP), were tested forinhibition and/or incorporation into an RNA product by the HCV RNAdependent RNA polymerase (RdRp). The incorporation reactions included 1mM of the phosphonate analog or ATP, 10 μM of [³³P]GpC, 10 μM of5′-AAAAAAAUGC-3′, and 2.5 μM of HCV RdRp in a buffer that contained 50mM Hepes, pH 7.3, 10 mM DTT, and 5 mM MgCl₂. The RNA products wereresolved on a 25% polyacrylamide-7M urea-TBE gel. As shown in FIG. 8,extension of [³³P]GpC by HCV RdRp was observed with ATP or 5′d-CH₂-ATP,but no extension with 5′d-ATP. The incorporation of 5′d-CH₂-ATP is lessefficient than that of ATP by 10-fold. To look for further elongationafter incorporation of ATP or 5′d-CH₂-ATP, the same experiment wasperformed in the presence of additional UTP (1 mM). Elongation productsby UTP were seen after ATP or 5′d-CH₂-ATP incorporation although thosefrom the latter were much weaker in intensity. This result clearlydemonstrates that HCV NS5B can incorporate a phosphonate nucleotide whenit is isosteric to the natural substrate.

[0150] It should be especially recognized that there have been very fewexamples of utilizing phosphonates by either DNA or RNA polymerases. Aphosphonate analog of AZT was shown to be very ineffective to HIVreverse transcriptase as both Km and kcat for incorporation areunfavorable by 3 orders of magnitude compared to those of AZT (Freeman,G. A. et al. J. Med. Chem. 1992, 35, 3192-3196). Phosphonate (5-deoxy)UTP was reported to be a pseudoterminator of RNA synthesis by E. coliRNA polymerase [Savochkina, L. P. et al. Mol. Biol. (Mosk) 1989, 23,1700-1710]. However, to the best of the inventor's knowledge, no studyhas yet been reported for successful phosphonates as potentialsubstrates or inhibitors for the HCV RNA-dependent RNA polymerase. Ourstudy clearly demonstrated, for the first time, that a phosphonateanalog of ATP may be utilized as a substrate by HCV RdRp. Interestingly,only that which is isosteric to ATP was incorporated by HCV RdRp to alesser degree than ATP, indicating that the polymerase requires specificalignment between the α-phosphate of the elongation nucleotide and the3′-OH of the preceding nucleotide for efficient phosphodiester bondformation.

[0151] Exemplary results of such tests are depicted in theautoradiograph of FIG. 8 showing RNA products from incorporations of ATPand phosphonate analogs of ATP by HCV RdRp. Each reaction contained 2.5μM of RdRp, 10 μM of [³³P]GpC, 10 μM template, and the followingnucleotides (1 mM): lane 1, none; lane 2, ATP; lane3,5′-deoxy-5′-phosphonate ATP; lane 4,5′-deoxy-5′-methylene phosphonateATP; lane 5, ATP and UTP; lane 6,5′-deoxy-5′-phosphonate ATP and UTP;lane 7,5′-deoxy-5′-methylene phosphonate ATP and UTP. The reactionproducts were resolved on a 25% polyacrylamide-7 M urea-TBE gel and thegel was scanned on a PhosporImager.

[0152] Thus, specific embodiments and applications of antiviralphosphonate compounds have been disclosed. It should be apparent,however, to those skilled in the art that many more modificationsbesides those already described are possible without departing from theinventive concepts herein. The inventive subject matter, therefore, isnot to be restricted except in the spirit of the appended claims.Moreover, in interpreting both the specification and the claims, allterms should be interpreted in the broadest possible manner consistentwith the context. In particular, the terms “comprises” and “comprising”should be interpreted as referring to elements, components, or steps ina non-exclusive manner, indicating that the referenced elements,components, or steps may be present, or utilized, or combined with otherelements, components, or steps that are not expressly referenced.

What is claimed is:
 1. A pharmaceutical composition comprising: anucleotide analog having a structure according to Formula 1 or Formula2, wherein the nucleotide analog is present in the composition effectiveto inhibit a viral polymerase of an HCV virus or to act as a substratefor the viral polymerase of the HCV virus;

wherein Z¹ and Z² are independently H, alkyl, halogen, OR⁵, SR⁵, NR⁵R⁶,CO-alkyl, CO-aryl, or CO-alkoxyalkyl, and wherein W is H, OR⁵, SR⁵,NR⁵R⁶, NH(NR⁵R⁶), N(alkyl)(NR⁵R⁶), CN, C(O)NR⁵R⁶, C(NH)NR⁵R⁶, orhalogen; wherein V is hydrogen, halogen, OR⁵, SR⁵, NR⁵R⁶, NH(NR⁵R⁶),N(alkyl)(NR⁵R⁶), CN, C(O)NR⁵R⁶, or C(NH)NR⁵R⁶, and wherein Y is H,alkyl, halogen, OR⁵, SR⁵, NR⁵R⁶, CO-alkyl, CO-aryl, or CO-alkoxyalkyl; Xis a covalent bond between the C4′-atom of the sugar and the carbon atomin the phosphonate group, O, CH₂, CHR⁵, CHHalogen, or C(Halogen)₂; D isCH₂, CHHalogen, or C(Halogen)₂; R¹ and R² are independently H,phosphate, or a group that is preferentially removed in a hepatocyte toyield a corresponding OH group; R₁′, R₂′, R₃′, and R₄′ are independentlyH or alkyl; R³ and R⁴ are H, or where at least one of R₁′, R₂′, R₃′, andR₄′ is alkyl, R³ and R⁴ are independently H, phosphate, acyl, alkyl, ora group that is preferentially removed in the hepatocyte to acorresponding C2′-OH group or C3′-OH group; R⁵ and R⁶ are independentlyH, alkyl, or acyl.
 2. The pharmaceutical composition of claim 1 whereinthe nucleotide analog has a structure according to Formula
 1. 3. Thepharmaceutical composition of claim 2 wherein X is a covalent bondbetween the C4′-atom of the sugar and the carbon atom in the phosphonategroup, O, or CH₂, and wherein at least one of R₁′, R₂′, R₃′, and R₄′ isCH₃.
 4. The pharmaceutical composition of claim 3 wherein R₂′ is CH₃. 5.The pharmaceutical composition of claim 3 wherein Z₁ and Z₂ is H, andwherein W is NR⁵R⁶.
 6. The pharmaceutical composition of claim 1 whereinthe nucleotide analog has a structure according to Formula
 2. 7. Thepharmaceutical composition of claim 6 wherein X is a covalent bondbetween the C4′-atom of the sugar and the carbon atom in the phosphonategroup, O, or CH₂, and wherein at least one of R₁′, R₂′, R₃′, and R₄′ isCH₃.
 8. The pharmaceutical composition of claim 7 wherein R₂′ is CH₃. 9.The pharmaceutical composition of claim 7 wherein Y is H or CH₃, andwherein V is OH, or NR⁵R⁶.
 10. The pharmaceutical composition of claim 1wherein the viral polymerase is disposed in a cell infected with HCV.11. The pharmaceutical composition of claim 10 wherein the cell isdisposed in a patient infected with HCV.
 12. A prodrug of the nucleotideanalog present in the pharmaceutical composition according to claim 1,wherein the prodrug includes a moiety that is preferentially removedfrom the prodrug in a hepatocyte.
 13. The prodrug of claim 12 whereinthe moiety is covalently bound to the phosphonate group and comprises anamino acid or forms a cyclic group with the phosphonate group.
 14. Apharmaceutically acceptable salt of the nucleotide analog present in thepharmaceutical composition according to claim 1.